Phylogeography and population structure of Lagocephalus spadiceus (Richardson, 1845) (Tetraodontiformes, Tetraodontidae) in the South China Sea

Abstract The climate fluctuations during the Late Pleistocene significantly influenced the phylogeographic structure and historical dynamics of marine fishes in the marginal seas of the western Pacific Ocean. The puffer fish, Lagocephalus spadiceus, holds substantial nutritional and economic value in the South China Sea. To investigate the demographic history and population structure of the L. spadiceus, the mitochondrial DNA COI and Cyt b gene datasets from 300 individuals across eight populations in the South China Sea were sequenced. Our findings revealed high haplotype diversity (0.874 ± 0.013) and low nucleotide diversity (0.00075 ± 0.00058). The phylogenetic tree and haplotype networks revealed no significant genetic differentiation along the northern coast of South China Sea. Neutrality tests, mismatch distribution analyses, and Bayesian skyline plots suggested that L. spadiceus underwent population expansion during the Late Pleistocene. Both ocean currents and climate change significantly influenced the geographical distribution and genetic population structure of L. spadiceus.


| INTRODUC TI ON
In marine species, the phylogeography and genetic differentiation were influenced by historical events, including ocean current systems, vicariance, Pleistocene climatic cycles, and life-history characteristics of organisms (Ding et al., 2018;Liu et al., 2007).It is worth noting that some marine fishes have fragile genetic structures due to their extensive larval and adult dispersal (Ashrafzadeh et al., 2021;Caccavo et al., 2018).Climate oscillations during the Pleistocene greatly altered the environment of marginal seas of the western Pacific, including the South China Sea (SCS).During glacial periods, the SCS formed a semi-enclosed sac-shaped gulf and exposed approximately 0.7 million km 2 of continental shelf (Wang & Sun, 1994).Previous molecular studies have shown that many marine fishes with high mobility exhibit low genetic structure in the SCS, such as Cirrhimuraena chinensis (Li et al., 2014) and Nuchequula mannusella (Gao et al., 2019).The phylogeographic study of marine fishes alive in the SCS has particular significance for interpreting the consequences of past events, geological configurations, and modern oceanographic aspects in this environment (He et al., 2010).
Lagocephalus spadiceus is a non-toxic Lagocephalus species (Tuney, 2016), belonging to Tetraodontiformes, Tetraodontidae, and Lagocephalus.It is a nearshore warm-water demersal fish that inhabits depths between 3 and 200 m (Tuncer et al., 2008), distributed along the southern coast of Africa in the Indian Ocean, eastward to Indonesia and the Philippines of the Pacific Ocean, and northward to the coast of China (Liu et al., 2016).In China, it occurs along the coastal areas of the SCS.Almost L. spadiceus is imported from China in Japan (Yamaguchi et al., 2013), but this also makes it an easy target for widespread exploitation.Recently, since the continuous increase in fishing intensity and deteriorating environmental conditions, the wild resources of L. spadiceus have been drastically reduced (Hardy et al., 2014).There are fewer reports on germplasm resources and genetic diversity evaluation of L. spadiceus.To better protect and rationally develop the wild germplasm resources of L. spadiceus, it is imperative to conduct a genetic diversity assessment to establish a theoretical basis for the scientific conservation and sustainable utilization of its genetic resources.
Genetic diversity is a vital component of biodiversity and a prerequisite for the continuous adaptation of species or populations to environmental change and survival evolution.Species with a higher genetic diversity possess a greater ability to environmental changes (Roldan et al., 2000).Mitochondrial DNA (mtDNA) constitutes a tiny fraction of organismal genome size but has been widely used as a marker of molecular diversity in animals for the past four decades (Galtier et al., 2009).This tool has been widely embraced by population geneticists, following the works of Avise et al. (1987) and Moritz et al. (1987), among others.Experimentally, mtDNA is present in most cells with high copy numbers and is relatively easy, rapid, and inexpensive to sequence (Zink & Barrowclough, 2008).Due to the relationship between the rate and time of evolution, effective information sites are different, and their resolving power is different.Therefore, concatenating COI and Cyt b markers can increase the number of effective genetic sites, resulting in more accurate and effective information compared to single gene analysis (Barrientos-Villalobos & Schmitter-Soto, 2019;Halasan et al., 2021).
Our study examines the phylogeography, population genetic diversity, and demographic history of L. spadiceus using COI and Cyt b gene datasets.As of current knowledge, there has not been a documented report on the population genetic of L. spadiceus.This study aims to address the lack of information on L. spadiceus in the region by providing a comprehensive background report.Meanwhile, our study has contributed to a better understanding of the evolutionary process that has influenced the phylogeography of coastal marine fishes in China.

| Sample collection
A total of 300 specimens of L. spadiceus were obtained from eight geographic locations, including Beihai (BH), Zhanjiang (ZJ), Leizhou (LZ), Danzhou (DZ), Dongfang (DF), Maoming (MM), Shanwei (SW), and Zhangzhou (ZZ) in the South China Sea (Figure 1, Table 1).All L. spadiceus specimens were collected through bottom trawl surveys from April 2022 to April 2023.These were specifically for the purpose of investigating the structure of fishery resources in the Beibu Gulf and its adjacent seawaters.A morphological analysis was used to identify species (head, dorsal surface, and ventral surface are covered with small spines, the dorsal side of the body is brownish-yellow or yellow-green, and caudal fin is white at the upper and lower tips) (Chen & Zhang, 2015;Liu et al., 2016).Fishes were given access to muscle tissue, which was then stored in 1.5 mL vials with 95% ethanol at −20°C until genomic extraction.The experimental animal care and use adhered strictly to the guidelines, policies, and laws concerning animal welfare as stipulated by the Animal Research and Ethics Committees of the Guilin University of Technology (Approval Code: GLUT-EAEC2021010) animal welfare laws, guidelines, and policies.

| DNA extraction, PCR amplification, and sequencing
The total DNA of L. spadiceus was extracted from each muscle following the FastPure Cell/Tissue DNA Isolation Mini Kit (Nanjing, China).The purity and concentration of DNA were checked using an ultra-microspectrophotometer (NanoDrop 2000, United States of America).The primers of mtDNA were adapted from Li et al. (2018).The COI was boosted by the primers COI-F: 5′-AAACCACCGCCTGACACTC-3′ and COI-R:

| Genetic structure
The Bayesian information criterion (BIC) in jModelTest v2.1.10Darriba et al., 2012) was used to establish a substitution model for the haplotype datasets before phylogenetic analysis.Subsequently, the mitochondrial COI and Cyt b gene dataset haplotypes were used to reconstruct the phylogenetic tree using the Bayesian inference (BI).The congeneric species Lagocephalus laevigatus was chosen as an outgroup, from the NCBI access number NC_015345.1 (COI and Cyt b).Bayesian inference study was carried out using MrBayes v3.2.7 (Ronquist et al., 2012), and one set of four chains was permitted to run concurrently for 20 million generations.Every 1000 generations, a sample of the tree was taken, with the first 25% being eliminated as burn-in.As the sampled generations increased, the log likelihood maintained a constant level, and stationarity was attained when the split average frequencies' average standard deviation was less than 0.01 (Hall, 2016).Phylogenetic tree editing was done with FigTree v1.4.4 (http:// tree.bio.ed.ac.uk/ softw are/ figtr ee/ ), and a median-joining haplotype network was produced using PopART v1.7 (Leigh & Bryant, 2015).
Genetic distance within and between populations was calculated using MEGA v7.0 (Kumar et al., 2016).Subsequently, AMOVA was used to quantify genetic variation using F-statistics at two geographically distinct levels of subdivision: among and within populations.To test for statistical significance, 10, 000 permutations of the fixation index F ST were performed between pairs of populations using Arlequin v3.5 (Excoffier & Lischer, 2010).

| Demographic history
The Tajima's D (Tajima, 1989) and Fu's Fs (Fu & Li, 1993) tests were utilized to check for neutral evolution.To examine population growth, the mismatch distribution (Rogers & Harpending, 1992) between the sum of squared deviations (SSD) and Harpending's raggedness index (Rg) was analyzed with Arlequin v3.5 (Excoffier & Lischer, 2010).Changes in effective population size (Ne) over time were deduced using Bayesian skyline plot analysis in BEAST v2.6.3 (Bouckaert et al., 2019).To account for possible site-specific variations, the rate of clock mutation was fixed at 1 × 10 −8 per year, as recommended for reef fishes (Delrieu-Trottin et al., 2017).With a sample every 1000 iterations, 100 million generations of separate independent Markov chain Monte Carlo (MCMC) studies were carried out.The molecular clock was calibrated using an average divergence rate of 2% per million years for mtDNA (Schubart et al., 1998).ESS values were detected until they reached 200, and these parameter values were displayed in Tracer v1.7.1 (Rambaut et al., 2018).
The correlation between genetic diversity and longitude and latitude of sampling locations showed that the haplotype diversity (r = .29)and nucleotide diversity (r = .31) of L. spadiceus populations tend to rise with latitude but did not change significantly with longitude (r = .08and .04,respectively) (Figure 3).

| Genetic structure and differentiation
The molecular evolution model with the gamma shape parameter (HKY+I+G) was found to be the best substitution model for the COI and Cyt b gene datasets by the jModelTest.From this result, a BI tree was built to determine phylogenetic relationships across populations (Figure S1).The BI tree was dispersed with haplotypes from each population and lacked well-supported groups.Neither significant genealogy branches nor haplotype clusters could be identified about the sampling locations.
Network analysis and the phylogenetic tree showed similar results.The connection between several haplotypes resembled a star, with certain prominent haplotypes like Hap_1, Hap_2, Hap_4, and Hap_22 (Figure 2).Unnoticeable clades in the network diagram of reticulations do not correlate with sampling locations, suggesting a substantial gene flow among populations and recent population expansion.It was found that relationships between populations were not linked to geological networks, but instead to the haplotypes that were present in each population.According to these results, there was no obvious phylogeographical pattern of L. spadiceus in the South China Sea.
The degree of genetic variation between populations was evaluated using F st pairwise comparisons.The F st values were typically low and even negative, as shown in Table 2; only the F st values between the LZ population and other populations were higher and significant (p < .05).The genetic distance between and within populations was at the same level, with little differentiation (Table 2).
The range under investigation had no significant genetic structure.
Hierarchical AMOVA analysis revealed that genetic variation in all populations existed within populations, while only a small proportion of genetic variation could be attributed to differences between populations (Table 3).

| Demographic history
A unimodal distribution was observed in the mismatch distribution analysis (Figure 4a), which was consistent with the expected distribution under a sudden expansion model (Rg = 0.0536, p > .05,Table 4).
It is also possible that populations had undergone expansion in the past (as indicated by the star-like networks).The sum of the squared deviation (SSD) showed that there was no significant deviation from the growth and expansion model (p > .05).The Tajima's D and Fu's Fs tests of L. spadiceus were significantly negative (Tajima's D = −1.905,p < .05;Fu's Fs = −10.543,p < .01,Table 4).Typically, such values indicated that L. spadiceus may have experienced population expansion.
Bayesian skyline plots supported demographic scenarios explaining the recent population expansion of L. spadiceus (Figure 4b).
The population experienced a significant increase, followed by a period of demographic stability.The calculated population expansion F I G U R E 2 Median-joining network for COI and Cyt b gene datasets haplotypes of Lagocephalus spadiceus.The size of the circles is proportional to haplotype frequency.
time is approximately from 0.025 to 0.010 Mya during the Late Pleistocene (Figure 4b).

| Genetic diversity
Haplotype diversity (H d ) and nucleotide diversity (π) are two important indicators to measure genetic diversity, and π represents the proportion of each haplotype in the populations, which can reveal the polymorphism of mtDNA in the populations more accurately (Chen et al., 2022).Grant and Bowen (1998) concluded that H d was higher than 0.5 and π was greater than 0.005, which indicated higher species diversity.This study revealed high levels of haplotype and low levels of nucleotide diversity, which is common among some marine fish species (Avise et al., 1987;Zhang et al., 2006).
High genetic diversity plays a crucial role in the exploitation and restoration of fishery resources (DeWoody et al., 2021).Assessing genetic diversity is an effective approach to the adaptability and survival ability of species in response to environmental changes (Schmitt & Hewitt, 2004), which is essential for species management and conservation.The fish of L. spadiceus is an economic species and has been caught for a long time.Compared with other marine fishes in the same sea area, L. spadiceus had a lower nucleotide diversity (Niu et al., 2019;Xu et al., 2021;Yi et al., 2021), reflecting that L. spadiceus population in the South China Sea has a fragile genetic diversity and requires conservation as well as a sustainable development planning from fishery management.

| Genetic structure and differentiation
Genetic distance is a crucial factor in determining the genetic relationship between species (Mather et al., 2017).Shaklee et al. (1982) proposed a classification of fish genetic distance at the population level (0.05), species level (0.30), and genus level (0.90).In this study, it was found that the genetic distance between populations was small, indicating a close genetic relationship between these populations.
According to the coalescent theory (Crandall & Templeton, 1993), more diverse populations have longer coalescence times and larger coalescent effective population sizes than less diverse populations (assuming the same mutation rate).Therefore, the ancestral haplotype was the most widely distributed.In the COI and Cyt b gene datasets, Hap_1 and Hap_2 were found to be the dominant haplotypes (Figure 2) and may be the origin of L. spadiceus.In the Bayesian skyline plots, the X-axis represents time using a mutation rate of 2% per million years ago (Mya) (Schubart et al., 1998), and the Yaxis represents the effective population size (Ne).The two approaches reveal obvious signals of demographic expansion for the South China Sea.The mean estimate and both 95% highest posterior density (HPD) limits are indicated.

TA B L E 4
Neutrality tests and mismatch distribution parameter estimates for L. spadiceus.
In the phylogenetic analysis, the haplotypes of eight populations were randomly distributed.The haplotype network and phylogenetic tree also showed no clear pedigree structure corresponding to geographical location.This pattern suggests that the species went through a bottleneck event followed by a population expansion (Grant & Bowen, 1998).The dispersal of larvae with ocean currents is an important cause of the limited genetic differentiation of marine fishes that have a geographically large distribution range (Strathmann et al., 2002).In this study, L. spadiceus were caught in the spring and autumn, during this time, the China Coastal Current and the South China Sea Warm Current flowed northward into the East China Sea (Figure 1) (Wang et al., 2015;Yang et al., 2008).Previous studies have reported that extensive gene exchange occurs over a wide geographical range in marine fishes (Grant & Bowen, 1998;Niu et al., 2019;Yi et al., 2021).F st is a significant measure in evaluating genetic diversity among populations (Holsinger & Weir, 2009).A higher F st value suggests a greater level of genetic differentiation.According to Wright (1951)   Many studies have demonstrated a weak genetic differentiation between the geographical populations of surface marine fish that can migrate long distances or swim.This can be attributed to the free dispersal of floating eggs, fish larvae, juveniles, and adults, as well as the absence of significant geographical obstacles in the open ocean environment.Consequently, gene exchange occurs extensively and widely among these marine fish populations (Canfield et al., 2022;Hewitt, 2000;Palumbi, 1994).However, it should be noted that L. spadiceus, being a benthic fish, does not exhibit a long-distance migration behavior according to its life history.Therefore, the observed panmixia among populations may be attributed to their early life habits.The active diffusion of fish larvae and juveniles as well as marine environmental factors, such as ocean circulation and climate change in the Late Pleistocene, have played crucial roles in shaping the systematic geographical pattern and population genetic structure of L. spadiceus.

| The choice of mtDNA
Surprisingly, despite the popularity of mtDNA as a marker in evolutionary studies, this assumption only relies on a handful of comparisons involving mostly vertebrate species (Nabholz et al., 2009).
Depending on the species, the mtDNA mutation rate was much higher or lower than the nuclear DNA (nuDNA) rate.For example, it is not always clonal, far from neutrally evolving and certainly not clock-like, and the ratio of mitochondrial to nuclear mutation rate varies widely among animals (Allio et al., 2017;Galtier et al., 2009).
Despite these long-acknowledged concerns, similar results were obtained in several studies that have employed mtDNA and nuDNA to investigate genetic structuring and demographic history in populations of marine fishes (Adams et al., 2006;Machado-Schiaffino et al., 2009;Mccusker & Bentzen, 2010;Vinas et al., 2010;Yang et al., 2022).

| CON CLUS IONS
In this study, we present the first exploration of the genetic struc- Yunrong Yan: Funding acquisition (equal); writing -review and editing (equal).Xiuguo Zhang: Funding acquisition (equal); project administration (equal).

F
Map of sampling geographic locations for Lagocephalus spadiceus.The map was downloaded from the National Geomatics Center of China with modifications using Arcgis10.7.In spring and summer, the China Coastal Current (blue arrow) and South China Sea Warm Current (red arrow) flow northward into the East China Sea(Wang et al., 2015).Note: BH, Beihai; ZJ, Zhanjiang; LZ, Leizhou; DZ, Danzhou; DF, Dongfang; MM, Maoming; SW, Shanwei; ZZ, Zhangzhou.TA B L E 1 Sampling locations and descriptive statistics of genetic diversity of L. spadiceus.

F
Mismatch distribution (a) and Bayesian skyline plots (b) for COI and Cyt b gene datasets of Lagocephalus spadiceus.

4. 3 |
Demographic historyThis study utilized the Tajima's D, Fu's F S tests, and mismatch analysis to suggest that a population expansion event of the L. spadiceus population may have occurred from 0.025 to 0.010 Mya in the Late Pleistocene.L. spadiceus is mainly distributed less than 50 m depth, and spawns in coastal habitats and shallow shorelines.Therefore, the L. spadiceus distribution is closely related to historical sea level fluctuations.During the Last Glacial Maximum of the Pleistocene, the northern South China Sea encompassed the Beibu Gulf, a segment of the South China continent.This region was connected to Hainan Island, while Taiwan Island was linked to mainland China.The entire South China Sea was separated from the Indian Ocean to form a semi-closed basin(Wang, 1990).The survival range of marine fish decreased sharply; therefore, the L. spadiceus population may have moved and survived in one or more glacial refuges during this period, such as the semi-closed South China Sea.In the Late Pleistocene, the glaciation began to disappear and L. spadiceus might have experienced rapid population expansion when favorable conditions occurred.
ture of L. spadiceus in the SCS.The mtDNA sequences analysis of specimens from the SCS revealed no significant genetic differentiation among sampling sites, with low F st values indicating genetic homogeneity, which probably reflected widespread and recent historical interconnections during the post-glaciation.Hainan Island and Leizhou Peninsula did not affect the gene flow of L. spadiceus in the SCS.In its demographic history, it experienced a low effective population size during the Quaternary period that increased sharply after the Last Glacial Maximum.The phylogeographic pattern of L. spadiceus may be attributed to past population expansion and long-distance larval dispersal facilitated by present-day ocean currents.Mitochondrial DNA (mtDNA) markers, which are maternally inherited, solely represent a singular evolutionary history.Consequently, they fail to provide an accurate representaion of the overall population structure.To enhance our understanding of the population structure of L. spadiceus, it is imperative to conduct further research utilizing more precise nuclear genetic markers such as microsatellites, single nucleotide polymorphisms, and other mtDNA genes.These markers enhance the efficacy of molecular data in substatiating phylogeographic hypotheses, offering supplenentary | 9 of 11 XU et al. insights into population structure.This information can subsequently guide the formulation of n effective management policy and contribute to the conservation of fish resources.Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); resources (equal); software (equal); validation (equal); writing -original draft (equal); writing -review and editing (equal).Liangliang Huang: Conceptualization (equal); formal analysis (equal); funding acquisition (equal); project administration (equal); resources (equal); supervision (equal); writing -review and editing (equal).Tao Chen: Formal analysis (equal); methodology (equal); supervision (equal); visualization (equal); writing -review and editing (equal).Caiguang Wang: Data curation (equal); investigation (equal).Zhiqiang Wu: Data curation (equal); funding acquisition (equal); supervision (equal).Yanan Cheng: Formal analysis (equal); software (equal).Qiongyuan Su: Data curation (equal); investigation (equal).Bin Kang: Formal analysis (equal); funding acquisition (equal); supervision (equal); writing -review and editing (equal).

Population code D N S H H d π K
Note: D, sampling date; N, sample size; S, number of polymorphic sites; H, number of haplotypes; H d , haplotype diversity (±SD); π, nucleotide diversity (±SD); and K, mean pairwise difference.
Plots and trendlines of haplotype diversity (H d ) and nucleotide diversity (π) with longitude (yellow line) and latitude (blue line).Genetic distance and differentiation between pair populations of L. spadiceus.